Artificial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–10 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.948548

Advances in preparation and characterization of chitosan nanoparticles for therapeutics Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by University of Sydney on 09/01/14 For personal use only.

Krushna Chandra Hembram1, Shashi Prabha2, Ramesh Chandra3, Bahar Ahmed2 & Surendra Nimesh1 1Department of Biotechnology, School of Life Sciences, Central University of Rajasthan, Dist-Ajmer, Rajasthan, India, 2Department of Pharmaceutical Chemistry, Jamia Hamdard University, New Delhi, India, and 3Department of Chemistry,

University of Delhi, Delhi, India

Abstract Context: Polymers have been largely explored for the preparation of nanoparticles due to ease of preparation and modification, large gene/drug loading capacity, and biocompatibility. Various methods have been adapted for the preparation and characterization of chitosan nanoparticles. Objective: Focus on the different methods of preparation and characterization of chitosan nanoparticles. Methods: Detailed literature survey has been done for the studies reporting various methods of preparation and characterization of chitosan nanoparticles. Results and conclusion: Published database suggests of several methods which have been developed for the preparation and characterization of chitosan nanoparticles as per the application.

easily pass through fine capillaries and reach tissue sinusoids. Nanoparticles can be defined as sub-microscopic, colloidal particles with at least one dimension less than 100 nm. First report on polymeric nanoparticles by Birrenbach and Speiser (1976) triggered explosive research toward design and development of novel nanoparticles based drug delivery vectors (Birrenbach and Speiser 1976). Nanoparticles have emerged as one of the most promising vectors with a plethora of applications in targeted drug and gene delivery. Owing to their sub-microscopic size, nanoparticles provide better targeting and tissue penetration (Peer et  al. 2007). Nanoparticles engineered from several natural/synthetic polymers including chitosan and polyethylenimine (PEI) have been widely explored either to deliver anticancer drugs or DNA/siRNA. These nanoparticles can broadly be subdivided into (i) nanospheres which are spherical particles where the drugs can either be entrapped inside the sphere or adsorbed on the outer surface or both, (ii) nanocapsules consist of an inner liquid core surrounded by a solid polymeric shell and the drugs can either be entrapped inside the core or adsorbed on the outer surface or both (Figure 1). Nanoparticles have also been observed in different other types of shapes such as cylinders, nanorods, nanotubes, cones, spheroids, etc. (Adeli et al. 2009). Chitosan, an aminoglucopyran, derivatized from naturally occurring chitin, is composed of randomly distributed N-acetylglucosamine and b-(1,4)-linked glucosamine residues. Chitosan is synthesized by N-deacetylation of chitin, which is a heteropolymer of randomly distributed N-acetylglucosamine and glucosamine residues with b-1,4linkage, in the presence of alkali (Figure 2). Derivatization of chitin, under controlled conditions, results in chitosan with degree of deacetylation (DDA) between 40% and 98% and the molecular weight (MW) between 5  104 Da and 2  106 Da (Hejazi and Amiji 2003). The physicochemical and biological properties of chitosan depend on DDA and degree of polymerization (DP), which also determines the

Keywords: chitosan, co-precipitation, ionotropic gelation, microemulsion, nanoparticles, polyelectrolyte complex

Introduction Development of an efficient drug delivery system with desired therapeutic effects has been a major area of focus in industry and academia. The physicochemical properties and molecular structure of the drug defines its fate in the body upon administration. However, non-availability of drug to the desired diseased site and deposition at nonspecific sites may lead to adverse and toxic side effects. Several strategies have been proposed, to fabricate a system which can effectively and specifically deliver drugs to diseased target sites. One of the seminal works suggests application of prodrugs, which are the derivatized analogues of the active drugs that can reach the target site and be cleaved enzymatically or chemically to reveal the active drug molecule. Later, particle based drug carriers were designed, to improve the bioavailability of drug at the desired site. The size of the particles remarkably influenced its physicochemical properties and made them suitable for systemic application, as they can

Correspondence: Surendra Nimesh, Department of Biotechnology, School of Life Sciences, Central University of Rajasthan, NH-8, Bandar Sindri, DistAjmer-305801, Rajasthan, India. E-mail: [email protected] (Received 21 May 2014; accepted 28 May 2014)

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Figure 1. Different types of nanoparticles (A) Nanospheres where drug is either entrapped in the polymer matrix or adsorbed onto surface or both. (B) Nanocapsules where drug is either entrapped inside the hollow capsule or adsorbed onto surface or both.

MW of polymer. Chemical structure elucidation suggests that chitosan possesses reactive hydroxyl and amino groups and is usually less crystalline than chitin. Since, chitosan possesses primary amino groups with a pKa value of 6.3, it can also be considered as a strong base. Further, the charge and physicochemical properties of chitosan are dictated by the pH, owing to the presence of amino groups (Yi et al. 2005). At pH above 6, chitosan amino groups become deprotonated; the polymer loses its charge and solubility. On the contrary, at lower pH, the amino groups get protonated and become positively charged, making chitosan a water-soluble cationic polyelectrolyte. This transition between solubility and insolubility occurs at its pKa value between pH 6 and 6.5. Hence, chitosan is readily soluble in mild acidic media, such as acetic acid, hydrochloric acid, and insoluble at neutral and alkaline pH values. Along with pH, the solubility of chitosan also depends on the DDA, MW, and ionic strength of the solution. Under physiological conditions, chitosan can easily be degraded either by lysozymes or by chitinases, which can be produced by the normal flora in the human intestine or exists in the blood (Aiba 1992, Zhang and Neau 2002, Escott and Adams 1995). At an optimal N/P ratio (ratio of nitrogen of amine in chitosan to phosphate of DNA), chitosan can efficiently condense DNA to form nanoparticles, with sizes compatible with cellular uptake while rendering protection against enzymatic nuclease degradation (Huang et  al. 2005). Owing to these properties, chitosan is considered as biodegradable, biocompatible, and non-immuno-

genic polymer and has been widely explored for drug/gene delivery both in industry and academia (Özgel and Akbuga 2006, MacLaughlin et  al. 1998a, Richardson et  al. 1999, Borchard 2001, Guliyeva et  al. 2006, Mansouri et  al. 2004, van der Lubben et al. 2001). Since, chitosan nanoparticles have found such an important position in arena of drug/gene delivery, it appears quite relevant to describe and compare various methods explored for preparation and characterization of chitosan nanoparticles. Herein, we provide an exhaustive account of strategies employed for the preparation and characterization of chitosan nanoparticles and advancements thereafter.

Methods for preparation of chitosan nanoparticles Several methods have been reported for fabrication of chitosan nanoparticles, depending on the type of application and the desired size requirements (Table I).

Polyelectrolyte complex method Polyelectrolyte complex (PEC) formulation takes place due to electrostatic interaction between anion and cation, followed by charge neutralization (Figure 3). Due to the charge neutralization, polyelectrolyte complex are self-assembled and it leads to the fall in hydrophilicity. Nanocomplexes formulated can be of varying sizes from 50 to 700 nm. These polyelectrolyte complexes are used as vehicles for delivery of proteins, peptides, drugs, and plasmid DNA. Sharma et al. (2012) prepared IgA-loaded chitosan-dextran nanoparticles for the delivery of vaccine. The polyplexes were prepared by complex coacervation (polyelectrolyte complex) technique and the nanoparticles generated were in the size range of 300–500 nm with zeta potential around  40– 50 mV. The group concluded that the nanocomplexes of chitosandextran could be developed as simple but effective delivery system. Another study was done by Nam and coworkers where low molecular weight water soluble chitosan (LMWSC) nanocarriers were developed by the similar methods for insulin delivery (Nam et  al. 2010). The nanoparticles developed by the above method reported size to be approximately 200 nm. Novel nanoparticles of heparin and chitosan were prepared by Liu and coworkers using polyelectrolyte complexation method following simple and milder conditions (Liu et al. 2007). Effects of pH, MW, and concentration were taken into consideration while observing the size and yield of nanoparticles. Lower pH and moderate MW favored more nanoparticles complexation.

Ionotropic gelation method

Figure 2. Schematic representation of preparation of chitosan from chitin by deacetylation.

Chitosan nanoparticles are engineered using an ionic cross-linker in the ionotropic gelation method (Janes et al. 2001). Generally, tripolyphosphate (TPP) is used as ionic cross-linker. The preparation method involves mixing of two aqueous phases, one containing polymer chitosan and the other one consisting of poly-anion TPP, to form complex coacervate (Pan et al. 2002). The formation process involves the addition of TPP solution to chitosan while under constant magnetic stirring at room temperature (Lopez-Leon

Preparation and characterization of chitosan nanoparticles  3 Table I. Methods for preparation of chitosan nanoparticles. S. No. Method Methodology

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1 2

Polyelectrolyte complex Inotropic gelation

3

Micro emulsion

4

Covalent cross-linking

5

Incorporation and incubation

6

Solvent evaporation

7

Coprecipitation

8

Complex-coacervation

Outcome

Electrostatic interaction between anion and cation followed by charge neutralization Nanoparticles preparation realized by using a cross-linking agent such as TPP Nanoparticles are formed in the aqueous core of reverse micelle by using cosurfactant and a cross-linking agent Nanoparticles prepared by covalent cross-linking between chitosan and cross-linking agents like PEG, glutaraldehyde, etc This method involves mixing of protein with chitosan followed by flush mixing with TPP leading to formation of nanoparticles This method consists of addition of chitosan to aqueous phase to form emulsion and precipitation, followed by evaporation to obtain nanoparticles It involves coprecipitation of chitosan solution, prepared in low pH acetic acid solution, by addition to high pH 8.5–9.0 solution such as ammonium hydroxide resulting in formation of highly monodisperse nanoparticles Nanoparticles are prepared via formation of coacervates between cationic chitosan and anionic polyanions, polymers or biomacromolecules

et al. 2005). It leads to a milky appearance as the formation of chitosan nanoparticles occurs; these nanoparticles can be stored at room temperature. The nanoparticles formed can have significant applications as a drug/gene carrier system for biomedical applications both in vitro and in vivo. Chitosan-TPP/vitamin C nanoparticles were prepared via ionotropic gelation between the positively charged amino groups of chitosan-TPP and the vitamin C, with constant magnetic stirring at room temperature for 1 h to promote cross-linking (Alishahi et  al. 2011). The nanoparticles were isolated using ultracentrifugation at 10,000 rpm for 30 min and the freeze-dried nanoparticles could be stored at 4°C for further use. Gulati et al. proposed intranasal delivery of chitosan nanoparticles for migraine therapy. They formulated and evaluated sumatriptan succinate-loaded chitosan nanoparticles for migraine therapy in order to improve its therapeutic effect and reduce dosing frequency. Ionic gelation method had been used for the formulation of nanoparticles. Alam et  al. formulated thymoquinone (TQ)-encapsulated chitosan nanoparticles for nose-to-brain targeting via ionic gelation method for the treatment of Alzheimer’s disease. A variable ratio of thymoquinone was incorporated in the chitosan solution prior to the formation of nanoparticles (Alam et al. 2012). Other groups have also used ionic gelation method to prepare chitosan nanoparticle carrying vitamin C, through the gastrointestinal tract and to induce nonspecific immunity system in Oncorhynchus (rainbow

Figure 3. Polyelectrolyte complex method: the nanoparticles formation takes place due to electrostatic interaction between anion (DNA) and cation (chitosan), followed by charge neutralization.

Nanoparticles of size 50–700 nm can be prepared Size of nanoparticles can be optimized as per requirement Nanoparticles of size below 100 nm can be prepared via this method Variable size nanoparticles can be prepared Small size nanoparticles of about 100–150 nm can be prepared Nanoparticles of size 50 to 300 nm can be prepared by this method Nanoparticles of size as low as 10 nm can be prepared Size of nanoparticles varies depending on the anionic coacervate used

trout). Chitosan nanoparticles were shown to be suitable to encapsulate vitamin C in nano size range and maintain the immune-inducing property of vitamin C. Some researchers have modified ionic gelation method to develop ampicillin trihydrate-loaded chitosan nanoparticles and evaluated their antimicrobial activity (Saha et al. 2010). Staphylococcus aureus was used to test the antimicrobial property of such nanoparticles. They proposed that the polymer and cross-linking reagent concentrations, and also, sonication time were rate-limiting factors for the development of the optimized nanoparticle formulations. The chitosan nanoparticles were capable of sustained delivery of ampicillin trihydrate. Another group also used the modified ionic gelation method to formulate dopamine loaded chitosan nanoparticles for the treatment of Parkinson’s disease (De Giglio et al. 2011). The formulation of nanoparticles adsorbing dopamine, the neurotransmitter, may help in the administration to the brain of Parkinson’s disease patients.

Microemulsion method Reverse micelles are thermodynamically more stable liquid mixtures of water, oil, and surfactant. Macroscopically, they are homogeneous and isotropic and are structured on a microscopic scale into aqueous and oil micro-domains separated by a surfactant rich film. Surfactants are amphiphilic molecules, which in the presence of water or organic solvent spontaneously form spherical or ellipsoidal aggregates (micelles). There are normal micelles, which exist in water at rather low concentration of organic solvents. Reverse micelles are formed in a large number of organic solvents such as hydrocarbons (e.g., hexane, octane, isooctane, or benzene), long chain alcohols, chloroform, diethyl ether, etc. including their mixtures (Bellocq et al. 1984). In the reverse micelles, the reaction takes place in the aqueous core of the reverse micellar droplets. Here the aqueous solutions of monomer, cross-linking agent, and other hydrophilic compounds remain in the aqueous core (host nanoreactor) of the reverse micelles (Figure 4). Polymerization reaction,

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Figure 4. Microemulsion method: in the reverse micelles, the reaction takes place in the aqueous core of the reverse micellar droplets. Here the aqueous solutions of monomer, cross-linking agent and other hydrophilic compounds remain in the aqueous core (host nanoreactor) of the reverse micelles. Polymerization reaction, which leads to the formation of the nanoparticles, takes place within these aqueous cores by a primary growth process.

which leads to the formation of the nanoparticles, takes place within these aqueous cores by a primary growth process. The reverse micellar droplets are of nanometer size; hence any polymerization reaction taking place in these droplets will produce polymers of nanometer size. The reverse micellar droplets consist of swollen aqueous core stabilized by a layer of surfactant molecules and dispersed in oil. The nucleation process in such a system is continuous and can occur throughout the duration of the polymerization reaction. The number of the polymer particles formed increases steadily with time, but their size remains constant. Maitra et  al. prepared chitosan nanoparticles by microemulsion technique by entrapment of chitosan in the aqueous core of the reverse micellar system followed by cross-linking with glutaraldehyde (Banerjee et  al. 2002). The chitosan nanoparticles were formed using a surfactant, for example, AOT (sodium bis (2-ethylhexyl) sulfosuccinate). Initially, the surfactant was dissolved in n-hexane, and then chitosan and glutaraldehyde were added to the surfactant/n-hexane mixture with continuous stirring at room temperature (Banerjee et  al. 2002). The free amine groups of chitosan were cross-linked with glutaraldehyde and the organic solvent was removed by evaporation under low pressure, followed by harvesting of cross-linked chitosan nanoparticles. Excess of surfactant was removed by precipitation with CaCl2 and then centrifuged. The size of the lyophilized nanoparticles was obtained to be less than 100 nm. Also, the size could be altered by varying the concentration of glutaraldehyde. You et  al. (2006) prepared chitosan-alginate core-shell nanoparticles using water-in-oil reverse microemulsion template. The study drew the conclusion that these biocompatible and biodegradable nanoparticles could be used to encapsulate plasmid DNA for gene delivery via cell endocytosis pathway.

In one of the authors’ study, chitosan nanoparticles were prepared by cross-linking with glutaraldehyde in an aqueous core of the reverse micellar droplets (Manchanda and Nimesh 2010). An optically clear solution was obtained on re-suspending these chitosan nanoparticles in aqueous buffer. In vitro pH-dependent release of the adsorbed oligonucleotides from nanoparticles showed that at basic pH the release of oligonucleotides was found higher as compared with neutral and acidic medium.

Covalent cross-linking method Chitosan have the ability to make covalent bonds with varying functional cross-linkers. This method of nanoparticles preparation involves formation of covalent bonds between chitosan chains with agents like polyethylene glycol (PEG), dicarboxylic acid, glutaraldehyde, or monofunctional agents like epichlorohydrin. Chitosan have amino groups which become protonated when added to an acidic aqueous solution and makes it soluble in it. But in other solutions it is not soluble which limits its utility. So, to make it soluble in water or other organic solvents chitosan can be modified by adding PEG or other cross-linking agent. Such attempts can be made by graft copolymerization of chitosan through chemical modifications with other polymers, such as PEG of different MW (Sugimoto et  al. 1998). PEGylation of chitosan through hydroxyl groups was first proposed by Gorochovceva and Makuška (2004). For PEGylated chitosan nanoparticle preparation, chitosan molecules are chemo-selectively modified at its C-6 position of its repeating units by PEG. By using phthalic anhydride the C-6 position amino groups are protected. Sodium hydride (NaH) is used to catalyze the etherification between the chitosan and PEG. These PEGylated chitosan nanoparticles are applicable for gene/ drug delivery.

Preparation and characterization of chitosan nanoparticles  5

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Incorporation and incubation method This method is generally adapted for the preparation of the chitosan nanoparticle for delivery of protein molecules. In the incorporation method, the protein is first premixed with chitosan solution, pH adjusted to 5.5 and temperature at 20°C (Gan and Wang 2007). Flush mixing of TPP to the protein-chitosan solution leads to the spontaneous formation of chitosan-protein nanoparticles, followed by gentle stirring for 60 min. During the incorporation process, protein molecules are entrapped/embedded in the chitosanprotein nano-matrix, with some protein molecules also being absorbed at the particle surface. On the contrary, in the incubation method, chitosan nanoparticles are formed first via TPP coacervation, followed by mixing with solutions containing protein at pre-determined concentrations. These solutions are gently stirred for 60 min to allow protein adsorption onto the nanoparticles to reach isothermal equilibrium. In this method, protein loading is solely via adsorption on the surface of nanoparticles. Moghaddam et  al. studied the mucoadhesion and permeation enhancing properties of thiolated chitosan (chitosan-glutathion) coated polyhydroxyethyl methacrylate nanoparticles. Here the work done by them was carried out by incubation and incorporation of different model macromolecules like isothiocyanate dextran. It was concluded that all nanoparticles prepared by thiolated chitosan with MW of medium size showed the most mucoadhesion and penetration enhancement properties (Moghaddam et al. 2009) .

Solvent evaporation method The method involves formation of emulsion of chitosan followed by solvent evaporation. In the initial step, chitosan solution is added in to the aqueous phase to form the emulsion. Then evaporation of polymer solvent results in the formation of nanospheres due to precipitation. The chitosan is added to ethanol and then to this solution pDNATris buffer is added with rapid pouring of ethanol under magnetic stirring. By applying reduced pressure the solvent is removed to yield nanoparticles. Zhang et  al. (2013) prepared cyclosporin-A loaded PEGylated chitosan-modified lipid based nanoparticles by using an emulsification/solvent evaporation method where the average size of nanoparticles was observed to be 89.4 nm. The encapsulation efficiency of the cyclosporine-A onto chitosan modified nanoparticles was found to be 69.22%. In another published study, Chadha et al. (2012) employed modified solvent evaporation method to fabricate nanoparticles in two size ranges of 126–139 nm and 151–181 nm. The nanoparticles of lectin/chitosan were loaded with hydrochlorothiazide (HCT) and later complexed with b-cyclodextrin (HCT-b-CD). Maximum entrapment efficiencies of 81.8  1.7% and 91.1  1.5% were obtained for HCT and HCT-b-CD loaded nanoparticles, respectively.

Coprecipitation method This method involves coprecipitation of chitosan solution, prepared in low pH acetic acid solution, by addition to high pH 8.5–9.0 solution such as ammonium hydroxide resulting in formation of highly monodisperse chitosan nanoparticles. In a published study, lactic acid-grafted chitosan nanoparticles

were engineered via coprecipitation process by addition of lactic acid grafted chitosan to ammonium hydroxide to form coacervate drops. The resultant nanoparticles were highly monodisperse with size ∼10 nm (Bhattarai et  al. 2006). In another study, Gregorio et  al. prepared magnetic nanoparticles coated with chitosan by coprecipitation method using different chitosan concentrations (Gregorio-Jauregui et  al. 2012). Chitosan and 6-mercaptopurine coated magnetite nanoparticles were recently prepared by this method and used as a drug delivery system (Dorniani et al. 2013).

Complex coacervation method Complex coacervation method has been used to prepare chitosan nanoparticles through the formation of coacervates between cationic chitosan and anionic polyanions, polymers or biomacromolecules. Hu et al. (2002) prepared chitosanpoly(acrylic acid) nanoparticles where the size was observed to depend on the ratio between the two polyelectrolytes. Chitosan/alginate nanoparticles have been developed to deliver bioactive molecules such as proteins and nucleic acids (Gazori et al. 2009). Zheng et al. (2007) prepared chitosan nanoparticles using chitosans varying in MW and DDA, quaternized chitosan and trimethylated chitosan oligomer to encapsulate plasmid DNA encoding green fluorescent protein (GFP) using the complex coacervation.

Characterization of chitosan nanoparticles To nurture better understanding of the mechanism of nanoparticles formation and its influence on the biological properties, characterization of nanoparticles is highly desirable. Chitosan nanoparticles have been well characterized for their physicochemical properties in plethora of reports. Herein, we provide a brief overview of studies detailing techniques involved in physicochemical characterization of chitosan nanoparticles (Table II).

Size of chitosan nanoparticles Several studies have witnessed, that smaller particles are better at transfecting cells (Mumper et al. 1995, Prabha et al. 2002). As per the requisite of targeted drug delivery, the smaller sized nanoparticles possess better penetration than the larger counterparts and are even capable of crossing capillaries and tissue sinusoids. Further, the functionalization of the nanoparticles surface also determine the size of nanoparticles, followed by formation of protein corona that surrounds the particle during in vivo administration and thereby the nanoparticles biological fate (Kabanov 1999). Experiments dealing with nanoparticle size determination are often complicated due to the polydispersity of samples. However, information pertinent to nanoparticles size can be obtained by using multiple but complimentary techniques, such as atomic force microscopy (AFM) or transmission electron microscopy (TEM) combined with dynamic light scattering (DLS). Utilization of two different methods, namely, AFM or TEM and DLS results in probable discrepancy in the determination of size of nanoparticles. DLS studies are done by suspending nanoparticles in water or buffer, which makes them fully hydrated; on the contrary AFM or

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Table II. Techniques for characterization of chitosan nanoparticles. S. No. Technique Underlying principle 1 2

UV-Vis spectroscopy 1H NMR spectroscopy

3 4

FT-IR spectroscopy Dynamic light scattering

5

Transmission electron microscopy

6

Atomic force microscopy

7

Nanoparticle tracking analysis

8

Scanning electron microscope

9

Laser Doppler velocimetry

Based on Lambert–Beer law Based on the nuclear magnetic resonance of electrons in the molecules Based on the stretching and bending of atoms in molecules Measures the temporal fluctuations of the scattered light due to the Brownian motion of the particles and calculates particles size on the basis of Stokes–Einstein equation Employs a beam of electron which transmits through the specimen and can be focused on an imaging device such as fluorescent screen and CCD camera Consists of a cantilever with a very fine tip that scans the surface of the sample and a laser tracing its movement generates 3D images A laser beam traces the Brownian movement of particles suspended in a liquid and records in video format Employs high energy beam of electrons to scan the sample surface Measures the velocity of the particles during electrophoresis and calculates zeta potential employing Smoluchowski approximation

Analysis Determination of DDA Determination of DDA Determination of DDA Determination of particles size and size distribution High resolution technique for determination of shape and size of nanoparticles Determination of size and surface morphology Determination of size Information about surface morphology and size of nanoparticles Determination of surface charge of nanoparticles

TEM studies are done by drying samples on a glass slide or copper grid surface. A limited number of nanoparticles are visualized using AFM or TEM, while, several thousands of particles are analyzed during DLS measurements. AFM or TEM provides size information which is more of qualitative in nature, whereas, that from DLS is more quantitative and a bigger picture of the whole sample. Hence, the utilization of two different but complementary techniques provides an overall estimation of nanoparticles size. However, determination of nanoparticles size under physiological conditions is an interesting and challenging area.

NaCl to 890  71.6 nm in 150 mM NaCl. In another study, we fabricated nanoparticles of chitosan crosslinked with glutaraldehyde using reverse micellar system and size estimation done using DLS (Manchanda and Nimesh 2010). The average nanoparticles size was found to be 102 nm with narrow size distribution having low polydispersity index (PDI) of 0.121. Recently, Lu et al. prepared chitosan-graft-polyethylenimine (CP)/DNA nanoparticles for gene therapy of osteoarthritis. Particle size as determined by DLS was correlated to the weight ratio of CP:DNA, where nanoparticles size decreased as CP content increased (Lu et al. 2014).

Dynamic light scattering

Transmission electron microscopy

Size of particles can be calculated by measuring (i) average intensity change as a function of angle (ii) change in polarization (iii) change in the wavelength or (iv) change in the average intensity (Chu 1974). DLS, known as quasi-elastic light scattering (QELS) or photon correlation spectroscopy (PCS), is based on the change in the average intensity phenomenon of light. In this method, a sample solution containing the particles is placed in the path of a monochromatic beam of light and the temporal fluctuations of the scattered light due to the Brownian motion of the particles is determined (Chu 1974, Gugliotta et  al. 2000). This technique involves calculation of particles size on the basis of Stokes–Einstein equation. The properties of nanoparticles are highly influenced by the surrounding environment, for instance the size distribution at physiological conditions may differ depending on whether in water or in dry state. In this regard, DLS seems to be the more suitable method as it provides measurements in physiological buffers or biological fluids such as blood plasma. In one of our study, size of chitosan nanoparticles was determined by complexing chitosan 92-10-5 (DDA-MWN/P ratio) with pDNA using DLS in various media (Nimesh et  al. 2010). The size of nanoparticles in double distilled water was found to be 243  12 nm, which increased with increasing salt concentration from 391  43.7 nm at 10 mM

The size and surface morphology of nanoparticles can be confirmed by visualization under the TEM. In TEM, a beam of high energy electrons irradiates a sample and the resulting image can be seen on a fluorescent screen. When an irradiated beam of electron passes through the sample, transmitted as well as diffracted beams are obtained. The image is formed by interference between the transmitted and the diffracted beams. The image can be focused on an imaging device such as fluorescent screen and charged coupled device (CCD) camera. This permits a very high resolution of the order of 2 Å. However, the sample should be thin, approximately 500 Å. The samples for TEM analysis can be prepared by simple deposition of dilute suspensions on carbon coated copper grids or can be done by using a negative staining material as uranyl acetate. In one of our study, chitosan nanoparticles were fabricated by crosslinking with glutaraldehyde in an aqueous core of the reverse micellar droplets and characterized for size by TEM along with DLS technique (Figure 5) (Manchanda and Nimesh 2010). The TEM revealed nanoparticles with average size of 90 nm with narrow size distribution. In another study chitosan/DNA nanoparticles were prepared through simple electrostatic interaction between the cationic chitosan and anionic DNA (Figure 6; Nimesh et al. 2012). TEM investigation of nanoparticles thus generated showed an average size of 200 nm.

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Preparation and characterization of chitosan nanoparticles  7 computer, enables the tip to maintain either a constant force or constant height above the sample. In the constant force mode, the piezo-electric transducer monitors real time height deviation. In the constant height mode, the deflection force on the sample is recorded. The primary purpose of these instruments is to quantitatively measure surface roughness with a nominal 5 nm lateral and 0.01 nm vertical resolution on all types of samples. An atomic force microscope is capable of imaging features from 0.25 nm to 80 mm. When scanning a sample with an AFM, a constant force is applied to the surface by the tip at the end of a cantilever. The force with the cantilever in the AFM is measured by two methods. In the first method, the deflection of the cantilever is directly measured. In the second method, the cantilever is vibrated and changes in the vibration properties are measured. In a published study, Almalik et  al. (2013) prepared chitosan nanoparticles via complexation with TPP anions, followed by coating with hyaluronic acid (HA). AFM studies facilitated to highlight the presence of HA corona on chitosan nanoparticles, along with estimation of the thickness to about 20–30 nm (in a dry state). Figure 5. Transmission electron microscopy image of chitosan nanoparticles crosslinked with glutaraldehyde. The average particle size is 90 nm. (Adapted from Manchanda and Nimesh 2010).

Atomic force microscopy AFM is employed to image the surface in atomic resolution and also to measure the force at nano-newton scale. An atomically sharp tip is scanned over a surface with feedback mechanisms that enable the piezo-electric scanners to maintain the tip at a constant force (to obtain height information), or height (to obtain force information) above the sample surface. Tips are made from Si3N4 or Si, and extended down from the end of a cantilever. The nanoscope AFM head employs an optical detection system in which the tip is attached to the underside of a reflective cantilever. A diode laser is focused onto the back of a reflective cantilever. As the tip scans the surface of the sample, moving up and down with the contour of the surface, the laser beam is deflected off the attached cantilever into a dual element photodiode. The photodetector measures the difference in light intensities between the upper and lower photodetectors, and then converts to voltage. Feedback from the photodiode difference signal, through software control from the

Nanoparticle tracking analysis The technique of nanoparticle tracking analysis (NTA) captures videos of nanoparticles moving under Brownian motion when illuminated by laser light in a liquid medium. Basic instrument setup consists of a specially designed laser illumination chamber placed under a microscope objective, where the nanoparticles suspended in the liquid sample passing through the laser beam path are observed by the instrument as small points of light under rapid Brownian motion. NTA tracks movement of single particles under Brownian motion; that overcomes the sensitivity biasness toward larger particles as in case of DLS due the intensity weighted DLS measurement (Lou et al. 2009). Recently, this technique has been employed for estimation of size of chitosan nanoparticles as a complementary study to DLS, where number-weighted distribution has been obtained along with estimation of concentration of nanoparticles. In a published study, Lien et  al. (2012) prepared nanoparticles of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting, followed by characterization by NTA. The size of the nanoparticles varied from ∼100 to 153 nm depending on formulation and was in well accordance with results obtained

Figure 6. Transmission electron image (TEM) of chitosan/DNA complexes. The average size of complexes is 200 nm. The image A is at lower magnification with the bar equal to 1000 nm and image B is at higher magnification with the bar equal to 200 nm. (Adapted from Nimesh et al. 2012).

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8  K. Hembram et al. from DLS studies. In another study, Ballarin-Gonzalez et al. (2013) engineered chitosan/siRNA nanoparticles at different N/P ratio by simple electrostatic interaction and characterized the generated nanoparticle using NTA technique. The nanoparticles size was found to depend on the N/P ratio and ranged between ∼126 and 154 nm. In a recent study, realtime NTA was used to evaluate the propensity of curcumincontaining chitosan nanoparticles to muco-adhere and release curcumin under simulated colon conditions (Chuah et  al. 2013). The size of nanoparticles suspended in PBS revealed that 82% of the nanoparticles were within the size range of 100–350 nm with the majority occurring at 100 nm (0.4  106 particles per ml) and the remaining mostly between 200 and 300 nm (0.27  106 particles per ml). Further, the snapshots from the video generated using NTA showed a clear difference in the number of nanoparticles as well as the distance between the particles.

Surface morphology of chitosan nanoparticles The interaction of nanoparticles has been observed to depend on the shape and surface morphology of nanoparticles. Surface morphology of nanoparticles has been reported to be observed employing TEM, AFM, and scanning electron microscope (SEM). Majority of studies report chitosan/DNA nanoparticles as spherical particles and some authors have reported a mixture of globular, toroids, rod-like particles (Danielsen et  al. 2004, MacLaughlin et  al. 1998b, KopingHoggard et al. 2001, Huang et al. 2005). This discrepancy in the surface of nanoparticles could be due to the variation in the complexation conditions or because of modifications introduced in the chitosan backbone.

Scanning electron microscopy SEM also employs a high energy beam of electrons to scan the surface of the sample. As in TEM, here also the samples are analyzed after drying but here the samples are also coated with a thin layer of gold or platinum. SEM provides a direct picture of the surface of nanoparticles. This technique also provides size information but it has been widely used for investigation of surface morphology. In a published study, nanoparticles of chitosan cross-linked with TPP loaded with salicylic acid and gentamicin were prepared by ionic gelation. SEM investigation of nanoparticles suggested that they were spherical in shape, and the average size was about 200 nm (Jingou et  al. 2011). In another study, chitosan/siRNA nanoparticles were prepared by mixing chitosan solution to siRNA by rapid pipetting and the size determination studies using environmental SEM suggested that they had a mean diameter of approximately 50 nm (Alameh et al. 2010).

Transmission electron microscopy TEM has also been used to investigate the surface morphology of chitosan nanoparticles. In one of the author’s publication, chitosan nanoparticles were prepared by crosslinking with glutaraldehyde in reverse microemulsion; TEM images revealed particles with spherical shape and a smooth surface distributed throughout the sample (Figure 3) (Manchanda and Nimesh 2010). The sample preparation methodology consisted of loading the sample solution on a formvar

(polyvinyl formal) coated copper grid, followed by air drying. The copper grids were coated with formvar by dropping 0.5% (w/v) solution of formvar in chloroform on the water (previously degassed) surface. A thin film was formed on the water surface, onto which several clean copper grids were placed, with the matty surface downward. After 2–3 s, the grids along with the film were lifted off by a piece of filter paper with forceps and air dried (Manchanda and Nimesh 2010). In another study, TEM studies of chitosan/DNA nanoparticles showed highly spherical structures with narrow size distribution (Figure 4; Nimesh et al. 2012).

Surface charge of chitosan nanoparticles Nanoparticles surface charge is reported as zeta potential, which is the measure of the magnitude of the repulsion or attraction between particles. When a particle is in a solution containing ions it is surrounded by an electrical double layer of ions and counterions. The potential that exists at the hydrodynamic boundary of the particle is known as the zeta potential. It is determined by measuring the velocity of the particles during electrophoresis using laser Doppler velocimetry. The magnitude of the zeta potential governs the potential stability of the colloidal system. If the particles in suspension have high negative or positive zeta potential values then they tend to repel each other and there is no tendency for aggregation. On the other hand, if the particles have low zeta potential values then there is no sufficient repulsive force and the particles tend to agglomerate. In one of the authors study, the effect of pH on the surface charge of the chitosan/DNA nanoparticles was investigated by measuring zeta potential on Zetasizer (Nimesh et al. 2010). The surface charge was found to depend on the pH of the suspension medium where higher positive charge was observed at lower pH. In another study, Zhao et al. determined the zeta potential of the chitosan nanoparticles prepared at various MW weights but at fixed N/P ratio. Zeta potential increased from  1 to  23 mV with the increase in MW, and with the increase in pH from 6.9 to 7.5, zeta potential decreased from 0 to  20 mV.

Conclusions From the publication database, it is quite evident that chitosan nanoparticles have potential to effectively deliver drug/genes to target sites. Depending upon the requirement chitosan has been functionalized to achieve targeted delivery of the payload. Several methods have evolved for the preparation of chitosan nanoparticles followed by characterization. Physicochemical characterization of nanoparticles in terms of size, surface morphology, and surface charge is highly desired, in order to develop nanoparticles that can deliver the payload effectively and repeatedly to target sites.­­

Acknowledgements The authors gratefully acknowledge the help rendered by Rajesh Ranjan (IRIC, University of Montreal, Montreal) for helping with literature access.

Preparation and characterization of chitosan nanoparticles  9

Declaration of interest

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The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by Science & Engineering Research Board (SERB), Department of Science and Technology (SERC Fast Track Proposals for Young Scientists Scheme), Government of India.

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